U.S. patent number 9,403,294 [Application Number 14/255,892] was granted by the patent office on 2016-08-02 for method of making a droplet-generating device.
This patent grant is currently assigned to Bio-Rad Laboratories, Inc.. The grantee listed for this patent is Bio-Rad Laboratories, Inc.. Invention is credited to Thomas H. Cauley, III.
United States Patent |
9,403,294 |
Cauley, III |
August 2, 2016 |
Method of making a droplet-generating device
Abstract
Method of making a droplet-generating device. In the method, a
first droplet-generating device may be produced. The first
droplet-generating device may include a molded portion created at
least in part with a mold and also may include a plurality of
droplet generators each formed at least in part by the molded
portion. A set of droplets may be generated with each of one or
more of the droplet generators. A property of at least one set of
generated droplets may be determined. The mold may be modified
based on the property. A second droplet-generating device may be
produced that includes a molded portion created at least in part
with the modified mold.
Inventors: |
Cauley, III; Thomas H.
(Pleasanton, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Bio-Rad Laboratories, Inc. |
Hercules |
CA |
US |
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Assignee: |
Bio-Rad Laboratories, Inc.
(Hercules, CA)
|
Family
ID: |
51728420 |
Appl.
No.: |
14/255,892 |
Filed: |
April 17, 2014 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20140312534 A1 |
Oct 23, 2014 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61813137 |
Apr 17, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01F
15/0238 (20130101); B29C 33/3842 (20130101); B01L
3/502707 (20130101); B01L 3/502784 (20130101); B01F
15/0258 (20130101); B01F 13/0062 (20130101); B01F
3/0807 (20130101); B29C 33/3835 (20130101); B01F
3/0861 (20130101); B01L 2300/0816 (20130101); B01L
2200/0673 (20130101); Y10T 29/49989 (20150115); B01L
2400/0487 (20130101); Y10T 29/49998 (20150115); Y10T
29/49982 (20150115); B29C 33/42 (20130101); Y10T
29/49764 (20150115); B01L 2300/0867 (20130101); B01L
3/00 (20130101); B01L 2200/0689 (20130101); Y10T
29/49771 (20150115) |
Current International
Class: |
B29C
33/38 (20060101); B29C 33/42 (20060101); B01L
3/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Blaine R. Copenheaver, Authorized Officer, Commissioner for
Patents, "International Search Report" in connection with related
PCT Patent Application No. PCT/US2014/034572, dated Aug. 22, 2014,
2 pages. cited by applicant .
Blaine R. Copenheaver, Authorized Officer, Commissioner for
Patents, "Written Opinion of the International Searching Authority"
in connection with related PCT Patent Application No.
PCT/US2014/034572, dated Aug. 22, 2014, 11 pages. cited by
applicant.
|
Primary Examiner: Hong; John C
Attorney, Agent or Firm: Kolisch Hartwell, P.C.
Parent Case Text
CROSS-REFERENCE TO PRIORITY APPLICATION
This application is based upon and claims the benefit under 35
U.S.C. .sctn.119(e) of U.S. Provisional Patent Application Ser. No.
61/813,137, filed Apr. 17, 2013, which is incorporated herein by
reference in its entirety for all purposes.
Claims
I claim:
1. A method of making a droplet-generating device, the method
comprising: producing a first droplet-generating device including a
molded portion created at least in part with a mold and also
including a plurality of droplet generators each formed at least in
part by the molded portion; generating a set of droplets with each
of one or more of the droplet generators; determining a property of
at least one set of generated droplets; modifying the mold based on
the property; and producing a second droplet-generating device
including a molded portion created at least in part with the
modified mold; wherein the mold defines a projecting portion
including a plurality of intersecting ridges that create a
plurality of intersecting grooves in the molded portion, and
wherein the step of modifying the mold includes a step of removing
material from the projecting portion of the mold.
2. The method of claim 1, wherein the step of determining a
property includes a step of determining an average number of copies
per droplet of a reference species in each set of generated
droplets.
3. The method of claim 2, wherein the reference species is a
nucleic acid target, and wherein only a subset of the droplets in
each set of generated droplets contain at least one copy of the
nucleic acid target.
4. The method of claim 1, wherein the property corresponds to a
volume of individual droplets of the at least one set.
5. The method of claim 1, wherein the step of generating a set of
droplets includes a step of generating a plurality of emulsions
with the plurality of droplet generators, with each emulsion
containing portions of a same sample.
6. The method of claim 1, wherein the step of producing a first
droplet-generating device includes a step of attaching a sealing
member to a surface of the molded portion of the first
droplet-generating device to form circumferentially-bounded
channels from the plurality of intersecting grooves.
7. The method of claim 1, wherein the step of generating a set of
droplets includes a step of generating a distinct set of droplets
with each of two or more of the droplet generators, and wherein the
step of determining a property includes a step of determining a
property of each set of droplets generated.
8. The method of claim 1, wherein the step of determining a
property includes a step of determining a property of a distinct
set of droplets generated by each of the plurality of droplet
generators.
9. The method of claim 1, wherein the first droplet-generating
device has a first plurality of droplet generators, wherein the
second droplet-generating device has a second plurality of droplet
generators arranged in one-to-one correspondence with the first
plurality of droplet generators, and wherein the step of modifying
the mold causes the second plurality of droplet generators to be
configured to generate a more uniform size of droplet than the
first plurality of droplet generators.
10. The method of claim 1, wherein the first droplet-generating
device has a first plurality of droplet generators, wherein the
second droplet-generating device has a second plurality of droplet
generators arranged in one-to-one correspondence with the first
plurality of droplet generators, and wherein the step of modifying
the mold causes at least one droplet generator of the second
plurality to be configured to generate a smaller size of droplet
than a corresponding droplet generator of the first plurality.
11. The method of claim 1, further comprising (a) generating a set
of droplets with each of one or more droplet generators of the
second droplet-generating device and (b) determining a property of
least one set of droplets generated with the second
droplet-generating device.
12. The method of claim 11, further comprising a step of modifying
the modified mold if the property of least one set of droplets
generated with the second droplet-generating device is outside a
tolerance.
13. The method of claim 1, wherein the projecting portion includes
a junction region having a junction where two or more of the ridges
meet one another, and wherein the step of removing material
includes a step of decreasing a height of the junction region at
one or more positions of the junction region.
14. The method of claim 1, wherein the step of modifying the mold
includes a step of changing a dimension of a region of the
projecting portion by less than one micrometer.
15. The method of claim 1, wherein the step of removing material is
performed at least in part with a beam of radiation.
16. The method of claim 15, wherein the step of removing material
is performed by removal of material from the mold with a laser.
17. A method of making a droplet-generating device, the method
comprising: producing a first droplet-generating device including a
molded portion created at least in part with a mold and defining a
plurality of grooves and also including a plurality of droplet
generators each formed in part by an intersection of three or more
of the plurality of grooves; generating a set of droplets with each
droplet generator; determining an average number of copies per
droplet of a nucleic acid target in each set of generated droplets;
modifying the mold based on the average number of copies determined
for one or more of the sets of generated droplets; and producing a
second droplet-generating device including a molded portion created
at least in part with the modified mold.
18. A method of making a droplet-generating device, the method
comprising: producing a first droplet-generating device including a
molded portion created at least in part with a mold and also
including a plurality of droplet generators each formed at least in
part by the molded portion; generating a set of droplets with each
of one or more of the droplet generators; determining a property of
at least one set of generated droplets; modifying the mold based on
the property; and producing a second droplet-generating device
including a molded portion created at least in part with the
modified mold; wherein the molded portion of the first
droplet-generating device includes a surface and a plurality of
grooves formed in the surface, wherein each of the plurality of
droplet generators includes a channel intersection at which grooves
of the plurality of grooves meet one another.
19. The method of claim 18, wherein the step of producing a first
droplet-generating device includes a step of attaching a sealing
member to the surface of the molded portion of the first
droplet-generating device to form circumferentially-bounded
channels from the plurality of grooves.
20. A method of making a droplet-generating device, the method
comprising: producing a first droplet-generating device including a
molded portion created at least in part with a mold and also
including a plurality of droplet generators each formed at least in
part by the molded portion; generating a set of droplets with each
of one or more of the droplet generators; determining a property of
at least one set of generated droplets; modifying the mold based on
the property; and producing a second droplet-generating device
including a molded portion created at least in part with the
modified mold; wherein the first droplet-generating device has a
first plurality of droplet generators, wherein the second
droplet-generating device has a second plurality of droplet
generators arranged in one-to-one correspondence with the first
plurality of droplet generators, and wherein the step of modifying
the mold causes the second plurality of droplet generators to be
configured to generate a more uniform size of droplet than the
first plurality of droplet generators.
21. A method of making a droplet-generating device, the method
comprising: producing a first droplet-generating device including a
molded portion created at least in part with a mold and also
including a plurality of droplet generators each formed at least in
part by the molded portion; generating a set of droplets with each
of one or more of the droplet generators; determining a property of
at least one set of generated droplets; modifying the mold based on
the property; and producing a second droplet-generating device
including a molded portion created at least in part with the
modified mold; wherein the first droplet-generating device has a
first plurality of droplet generators, wherein the second
droplet-generating device has a second plurality of droplet
generators arranged in one-to-one correspondence with the first
plurality of droplet generators, and wherein the step of modifying
the mold causes at least one droplet generator of the second
plurality to be configured to generate a smaller size of droplet
than a corresponding droplet generator of the first plurality.
22. A method of making a droplet-generating device, the method
comprising: producing a first droplet-generating device including a
molded portion created at least in part with a mold and also
including a plurality of droplet generators each formed at least in
part by the molded portion; generating a set of droplets with each
of one or more of the droplet generators; determining a property of
at least one set of generated droplets; modifying the mold based on
the property; producing a second droplet-generating device
including a molded portion created at least in part with the
modified mold; generating a set of droplets with each of one or
more droplet generators of the second droplet-generating device;
and determining a property of least one set of droplets generated
with the second droplet-generating device.
23. The method of claim 22, further comprising a step of modifying
the modified mold if the property of least one set of droplets
generated with the second droplet-generating device is outside a
tolerance.
24. A method of making a droplet-generating device, the method
comprising: producing a first droplet-generating device including a
molded portion created at least in part with a mold and also
including a plurality of droplet generators each formed at least in
part by the molded portion; generating a set of droplets with each
of one or more of the droplet generators; determining a property of
at least one set of generated droplets; modifying the mold based on
the property; and producing a second droplet-generating device
including a molded portion created at least in part with the
modified mold; wherein the mold defines a projecting portion
including a plurality of intersecting ridges that create a
plurality of intersecting grooves in the molded portion, wherein
the step of modifying the mold includes a step of changing a
dimension of a region of the projecting portion, and wherein the
dimension is changed by less than one micrometer.
25. A method of making a droplet-generating device, the method
comprising: producing a first droplet-generating device including a
molded portion created at least in part with a mold and also
including a plurality of droplet generators each formed at least in
part by the molded portion; generating a set of droplets with each
of one or more of the droplet generators; determining a property of
at least one set of generated droplets; modifying the mold based on
the property; and producing a second droplet-generating device
including a molded portion created at least in part with the
modified mold; wherein the step of modifying the mold is performed
at least in part with a beam of radiation.
26. The method of claim 25, wherein the step of modifying the mold
is performed by removal of material from the mold with a laser.
Description
CROSS-REFERENCES TO OTHER MATERIALS
This application incorporates by reference in their entireties for
all purposes the following materials: U.S. Pat. No. 7,041,481,
issued May 9, 2006; U.S. Patent Application Publication No.
2010/0173394 A1, published Jul. 8, 2010; U.S. Patent Application
Publication No. 2011/0217712 A1, published Sep. 8, 2011; U.S.
Patent Application Publication No. 2012/0152369 A1, published Jun.
21, 2012; U.S. Patent Application Publication No. 2012/0190032,
published Jul. 26, 2012; U.S. Patent Application Publication No.
2012/0194805 A1, published Aug. 2, 2012; U.S. Patent Application
Publication No. 2013/0269452 A1, published Oct. 17, 2013; U.S.
Patent Application Publication No. 2014/0024023 A1, published Jan.
23, 2014; U.S. Patent Application Publication No. 2014/0080226 A1,
published Mar. 20, 2014; U.S. patent application Ser. No.
14/171,754, filed Feb. 3, 2014; U.S. patent application Ser. No.
14/171,761, filed Feb. 3, 2014; and Joseph R. Lakowicz, PRINCIPLES
OF FLUORESCENCE SPECTROSCOPY (2.sup.nd Ed. 1999).
INTRODUCTION
A microfluidic device can be designed to provide one or more
droplet generators. Each droplet generator can generate a set of
droplets from a sample-containing fluid and a carrier fluid. The
droplet generator may enclose partitions of the sample-containing
fluid with the carrier fluid to form an emulsion composed of sample
droplets in a continuous carrier phase.
The droplet generators of the microfluidic device can be designed
to generate a set of emulsions each containing monodisperse
droplets of the same nominal size. However, the actual size of
droplets generated by at least one of the droplet generators may be
unacceptably larger or smaller than the nominal size.
SUMMARY
The present disclosure provides a method of making a
droplet-generating device. In the method, a first
droplet-generating device may be produced. The first
droplet-generating device may include a molded portion created at
least in part with a mold and also may include a plurality of
droplet generators each formed at least in part by the molded
portion. A set of droplets may be generated with each of one or
more of the droplet generators. A property of at least one set of
generated droplets may be determined. The mold may be modified
based on the property. A second droplet-generating device may be
produced that includes a molded portion created at least in part
with the modified mold.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic flowchart of an exemplary method of making a
droplet-generating device, in accordance with aspects of the
present disclosure.
FIG. 2 is an exploded view of an exemplary droplet-generating
device produced with the method of FIG. 1 and including a plurality
of emulsion production units, in accordance with aspects of the
present disclosure.
FIG. 3 is a sectional view of the droplet-generating device of FIG.
2 taken generally along line 3-3 of FIG. 2 through one of the
emulsion production units of the device.
FIG. 4 is a bottom view of a molded portion of the
droplet-generating device of FIG. 2.
FIG. 5 is a fragmentary bottom view of the molded portion of FIG. 4
showing a recessed region of a microfluidic layer of one of the
emulsion production units.
FIG. 6 is a sectional view of a mold for creating the molded
portion of FIG. 4, with the mold sectioned to match FIG. 3, in
accordance with aspects of the present disclosure.
FIG. 7 is a view of a bottom component of the mold of FIG. 6.
FIG. 8 is a fragmentary view of the bottom component of FIG. 7,
taken generally at the region indicated at "8" in FIG. 7, with the
region including a set of intersecting ridges that shape
counterpart channels for the emulsion production unit of FIG.
5.
FIG. 9 is a fragmentary plan view of the bottom component of FIG.
7, taken generally at the region indicated at "8" in FIG. 7 around
a junction region that includes a junction where the intersecting
ridges of FIG. 8 meet one another.
FIG. 10 is a sectional view of the bottom component of FIG. 7,
taken generally along line 10-10 of FIG. 9.
FIG. 11 is a graph of height data collected from one of the
intersecting ridges in the junction region of FIG. 9, along a line
extending through points identified by a filled triangle and an
open triangle in FIG. 9, after removal of 0.5 micron of material
from an area of the junction region that is bounded in FIG. 9 with
a dashed rectangle, in accordance with aspects of the present
disclosure.
FIG. 12 is a graph of a concentration determined for a nucleic acid
target from each of eight sets of droplets generated by the eight
droplet generators of a working model of the droplet-generating
device of FIG. 2, with the working model having a molded portion
created with the mold of FIGS. 6 and 7 after selective modification
of two projecting portions of the mold in a manner illustrated in
FIGS. 9 and 10.
FIG. 13 is a graph of theoretical and experimentally determined
changes in the concentration of the nucleic acid target for droplet
generators "3" and "5" of FIG. 12.
FIG. 14 is a schematic view of a droplet-generating device having a
plurality of droplet generators that operate in parallel to
generate droplets of the same emulsion, in accordance with aspects
of the present disclosure.
DETAILED DESCRIPTION
The present disclosure provides a method of making a
droplet-generating device. In the method, a first
droplet-generating device may be produced. The first
droplet-generating device may include a molded portion created at
least in part with a mold and also may include a plurality of
droplet generators each formed at least in part by the molded
portion. A set of droplets may be generated with each of one or
more of the droplet generators. A property of at least one set of
generated droplets may be determined. The mold may be modified
based on the property. A second droplet-generating device may be
produced that includes a molded portion created at least in part
with the modified mold.
For some applications it is desirable to form emulsions comprising
droplets that are substantially monodisperse in size. The size of
droplets in an emulsion may be affected by several factors,
examples of which are the geometry of the droplet generator; the
sizes of the geometrical features of the droplet generator; the
physical properties (density, viscosity, and the like) of the
sample and carrier fluids; the presence or absence of surfactants
and/or other additives that affect interfacial energies; the flow
rates or velocities of the sample and carrier fluids; and the
properties of the materials that are used to construct the droplet
generator.
A droplet-generating device may be an at least partially molded
device comprising a plurality of droplet generators, such at least
2, 4, 6, 8, 16, 24, 32, or 96 droplet generators, among others. The
droplet-generating device may include microfluidic channels each
having a transverse dimension of less than one millimeter.
It may be desirable that the sizes of droplets from each of a
plurality of droplet generators are substantially the same. One
means of obtaining consistent droplet sizes among a plurality of
droplet generators is to replicate a droplet generator design
throughout the plurality. A limitation of this approach is that it
may be difficult to replicate geometric features across a plurality
of droplet generators with sufficient fidelity to ensure that
droplet size remains consistent among droplet generators. The
ability to replicate features may be limited by machining
tolerances of a mold or the like. To achieve consistent droplet
sizes among droplet generators it may be necessary to achieve
manufacturing tolerances on the order of one micron or less.
The methods disclosed herein may, for example, be used to tune the
uniformity of droplet sizes between or among a plurality of droplet
generators of a droplet-generating device that is molded at least
in part with a mold. The same working model of the mold (or at
least a component thereof) may be refined in one or more iterations
and used to mold successive copies (i.e., successive generations)
of the droplet-generating device (and droplet generators). In some
embodiments, the methods may be used to reduce droplet size
variations from a nominal droplet size between successive
generations of at least one of the droplet generators. Emulsions
produced by droplet generators each created at least in part with
the mold may have a more consistent and comparable size of
droplets. Assay results obtained from the emulsions may be directly
compared to one another with greater confidence and smaller assay
variation.
Further aspects of the present disclosure are described in the
following sections: (I) overview of methods, and (II) examples.
I. Overview of Methods
This section describes exemplary methods of making a
droplet-generating device, tuning a working model of a mold for the
device, and generating sets of emulsions with successive
generations of the droplet-generating device produced with the
working model before and after the working model is modified. The
method steps described in this section and elsewhere in the present
disclosure may be performed in any suitable order, in any suitable
combination, and each may be performed any suitable number of
times. Also, features of physical elements (e.g., the mold(s) and
device(s)) involved in any method step may be constructed as
described in this section and elsewhere in the present disclosure
(e.g., Section II).
FIG. 1 shows a schematic flowchart of an exemplary method 50 of
tuning a mold 52 to make a droplet-generating device 54. To
simplify the presentation, only fragmentary parts of a
channel-shaping component 56 of the mold are shown, both before
(mold 52 at the top) and after (mold 52' at the bottom)
modification of a working model (i.e., the same instance) of
channel-shaping component 56 to form modified component 56'. Also,
FIG. 1 shows only fragmentary parts of a molded portion 58 of
device 54 that are shaped by, and complementary to, regions of
channel-shaping component 56.
Channel-shaping component 56 may include a base 60 and a plurality
of projecting portions 62, e.g., projecting portions 62a-62c, that
project from a surface 64 (e.g., a planar surface) of the base.
Each projecting portion 62a-62c may include a ridge structure 66
formed by a plurality of ridges 68 and a junction 70 where the
ridges meet one another (intersect). Each projecting portion
(and/or ridge structure thereof) may include any suitable number of
ridges that intersect at the junction, such as at least three,
four, or more.
FIG. 1 depicts an artificially truncated version of each projecting
portion 62a-62c, namely, only a junction region 71 of the
projecting portion. The junction region may include junction 70 and
any suitable longitudinal portion of each ridge 68 extending from
junction 70. In FIG. 1, each ridge 68 is shown as truncated
(compare with FIG. 8) to more clearly illustrate the
three-dimensional structure of the junction region.
A first copy of a droplet-generating device 54 may be produced,
indicated at 72, at least in part with mold 52. Each ridge
structure 66 of mold 52 may define a corresponding open channel
structure 74 (interchangeably termed a groove structure or a
recessed structure) of molded portion 58, when the molded portion
is creating by a molding process with mold 52. (Only a truncated
portion of open channel structure 74 is shown to match the
truncated ridge structure shown for mold 52.) Each channel
structure may include a plurality of grooves 76 (interchangeably
termed open channels) and an intersection 78 where the grooves meet
one another. Each open channel structure may be recessed from a
surface 80 of molded portion 58 (e.g., a surface formed on a top
side or a bottom side of the molded portion). Surface 80 of molded
portion 58 may be defined during the molding process by surface 64
of mold component 56 and may be planar.
A sealing member may be attached to surface 80 of molded portion
58, to form a fluidic seal with the surface, as part of the step of
producing device 54 (see Example 1). The sealing member may
complete a microfluidic layer of the device by forming closed
(circumferentially-bounded) channels from grooves 76, and a droplet
generator 82 (e.g., droplet generators 82a-82c) at or near the
intersection where each set of channels meet one another.
An emulsion production unit may comprise a droplet generator, one
or more microfluidic channels, and sources of sample and carrier
fluid. The droplet generator may be used to contact sample and
carrier fluids to form an emulsion and may comprise an intersection
of microfluidic channels. The droplet generator may be configured
as a "tee" (an intersection of three channels), a "cross" (an
intersection of four channels), or other geometries useful for
generation of droplets. At least one of the channels may be in
fluid communication with a supply of sample fluid (e.g., an aqueous
sample fluid). At least one of the channels may be in fluid
communication with a supply of carrier fluid (e.g., oil). The
droplet generator may be capable of contacting sample fluid in one
or more of its channels with carrier fluid in one or more of its
channels, with the contact occurring at or near the intersection of
the channels, to form an emulsion composed of the carrier fluid as
a continuous phase and the sample fluid as a dispersed phase.
Droplets 84a-84c may be generated, indicated at 86, from the
corresponding droplet generators 82a-82c of device 54, to form a
set of emulsions 88a-88c. The emulsions may be formed in parallel
or serially, among others.
Droplets or emulsions may be generated by contacting a sample fluid
(interchangeably termed a sample-containing fluid) with an
immiscible carrier fluid. The sample fluid or the carrier fluid or
both may comprise at least one surfactant to stabilize the
emulsion. The emulsion may comprise droplets of the sample fluid
enveloped in the carrier fluid. The droplets may (or may not) be
substantially spherical. The size of the droplets may be
characterized by a diameter, a volume, or the like. Examples of
desirable droplet sizes include: microliter range (diameters on the
order of 1 millimeter); nanoliter range (diameters on the order of
100 microns); picoliter range (diameters on the order of 10
microns); femtoliter range (diameters on the order of 1 micron);
and the like.
Projecting portions 62a-62c of mold 52 may be formed as replicates
of one another, to produce droplet generators 82a-82c that each
generate droplets of substantially the same size. For example, the
projecting portions may be created by selectively removing material
from a surface of the mold with a mill, to recess a portion of the
surface that becomes surface 64, while selectively leaving behind
regions that form the ridges. An exemplary mill that is suitable is
a diamond mill (e.g., a single point diamond mill) capable of
milling with a precision of less than about 5, 2, or 1 micrometer,
among others.
However, this milling precision may be insufficient to create
droplet generators 82a-82c capable of generating the desired degree
of identity of droplet sizes relative to each other. For example,
the droplet size generated by the droplet generators may vary among
the droplet generators over an undesirably large range, such as
greater than about 1% or 2%, among others. In the schematic
representation of FIG. 1, projecting portion 62b is shown as being
different in size than projecting portions 62a and 62c of mold 52,
which may result in production of a droplet generator 82b of device
54 that generates droplets 84b than are unacceptably larger than
droplets 84a and 84c of droplet generators 82a and 82c,
respectively. Tuning mold 52 can reduce the difference in droplet
sizes from the droplet generators. (Any difference in size and/or
shape of any region of one projecting portion 62b relative to
projecting portions 62a and 62c may produce a difference in droplet
size generated by the corresponding droplet generators.) The mold
may be tuned such that the droplet generators each generate
droplets to within 2%, 1%, or 0.5%, among others, of a desired
volume.
One or more droplets from the set of droplets (i.e., the emulsion)
generated by each droplet generator may be tested (e.g., measured),
indicated at 90, to determine a property 92 of each set of
droplets. (The property is shown schematically as the diameter of
each set of droplets.) The property may correspond to the size
(e.g., the volume) of droplets of each emulsion and may be an
absolute or relative indicator of size.
Droplet sizes may be determined by any approach known in the art.
An example of an approach for determining droplet size is
microscopic and comparison with a size reference. Another exemplary
approach is to label the droplets with, for example, a dye, and
then to measure the amount of dye by an optical method such as
fluorescence. Another exemplary approach is to measure light
scattering from the droplets.
Still another exemplary approach is to determine an average level
(per droplet) of a reference species in each set of droplets (i.e.,
each emulsion). The droplets of each emulsion may be generated with
the reference species present at partial occupancy in the droplets.
The term "partial occupancy" means that only a subset of the
droplets of the emulsion contain at least one copy of the reference
species, while the remaining droplets of the emulsion do not
contain any copies of the reference species. Copies of the
reference species may be randomly distributed among the droplets of
the emulsion. The presence or absence of the reference species may
be detected for each of a plurality of individual droplets of the
emulsion. The level of the reference species then may be calculated
based on a number of the droplets that are positive (or negative)
for the reference species, out of the total (i.e., the fraction of
droplets that are positive or the fraction of droplets that are
negative for the reference species). The level may, for example, be
determined with Poisson statistics, may be described as a
concentration, and may be expressed as the average number of copies
of the species per droplet in the emulsion.
The reference species may have a known concentration in a sample
from which the droplets of an emulsion are formed. The volume of
droplets in the emulsion may be inferred based on the known
concentration and the level of the reference species determined for
droplets of an emulsion. As a simplified example, intended only for
illustration, the reference species may be known to be present in a
sample at 1000 copies per microliter (1 copy per nanoliter).
Droplets formed with portions of the sample may be determined to
have an average of 1.0 copy per droplet from a first droplet
generator and 1.1 copy per droplet from a second droplet generator.
Therefore, the first droplet generator generated droplets having a
volume of 1.0 nanoliter and droplets from the second droplet
generator generated droplets having a volume of 1.1 nanoliter. In
other cases, the determined level of the reference species can be
used directly for comparing droplet sizes from distinct droplet
generators, without knowing the concentration per unit volume of
the reference species in the sample.
The reference species may be a nucleic acid target (interchangeably
termed a target sequence). Detection of the target may include
amplifying the target in droplets of the emulsion, and detecting
signals from droplets to determine whether or not amplification of
the target occurred on a droplet-by-droplet basis. A concentration
(e.g., an average number of copies per droplet) of the reference
species can be determined from each emulsion formed with droplet
generators 82a-82c.
The property determined for each of one or more sets of droplets
(or from each set of droplets) may be compared to a tolerance, to
determine whether the property falls within or outside the
tolerance, indicated at 94. If the property determined for each set
falls within the limit of tolerance, the mold meets specification
and may be used to produce additional copies of the molded portion
of the droplet generating device, without further mold
modification, indicated at 96 and 98. If, however, the property
determined for each set is not within the limit of tolerance, the
mold does not meet specification and may be modified (tuned) to
improve its performance (or discarded if the mold cannot be tuned
readily).
The tolerance may be defined by a single value (e.g., a threshold
value) or a range of values (e.g., a range defined by a maximum
tolerated value and a minimum tolerated value), among others. For
example, the tolerance may represent a maximum size of droplet that
is acceptable for any droplet generator of the device, a maximum to
minimize acceptable size range for any droplet generator of the
device, or a maximum tolerated variation of droplet sizes among the
droplet generators of the device, among others.
A suitable modification of the mold may be determined, indicated at
100, based on the property determined for at least one set of
generated droplets. Mold 52 then may be modified, indicated at 102,
to form a modified mold 52' including a modified channel-shaping
component 56'.
The degree/type of modification needed for each projecting portion
62a-62c may be determined based on the property determined for each
corresponding set of droplets. The mold generally is modified only
where the performance of a counterpart droplet generator needs to
be improved. For example, in FIG. 1, droplets 84a and 84c each fall
within the tolerated size range, so corresponding projecting
portions 62a and 62c of mold 52 do not need to be modified further.
In contrast, droplets 84b are determined to be larger than the
maximum tolerated size, so corresponding projecting portion 62b of
mold 52 needs to be modified to decrease the size of droplets
generated by droplet generator 82b (in a new copy (the next
generation) of device 54).
The degree/type of modification needed for projecting portion 62b
may be determined based on the difference between the actual value
determined for the property of droplets 88b and a target value (a
preferred or nominal value) for the property. As an example, an
amount of material to remove from (or add to) junction region 71 of
projecting portion 62b may be calculated based on the magnitude of
the difference between the actual and target values (see Example
3). The material may be removed from (or added to) one or more
ridges adjacent the junction and/or the junction itself. In
exemplary embodiments, material may be removed from (or added to)
at least one carrier ridge (corresponding to a carrier channel), a
droplet ridge (corresponding to a droplet channel that carries
droplets away from the droplet generator), and from the junction.
The material may be removed from (or added to) the top and/or a
lateral side(s) of any ridge and/or the junction. Accordingly,
material removal/addition may alter a height and/or a width of a
region of one or more ridges and/or of the junction.
Material removal may performed by any suitable procedure. Exemplary
processing steps for material removal may include mechanical
machining, micromachining, laser machining, chemical etching, or a
combination thereof, among others.
Material may be added by any suitable procedure. Exemplary
processing steps for material addition may include forming a mask
(e.g., with photoresist or plasma) that selectively masks only a
region of the mold, and adding material to another region of the
mold that is not masked. Material may, for example, be added to the
non-masked region of the mold by chemical/physical vapor
deposition, sputtering, or the like. The mask then may (or may not)
be removed from the masked region before the modified mold is used
in a molding process.
One or more processing steps for mold modification may be applied
selectively to some or all of the plurality of replicated features
within the mold to increase uniformity. For example, if a first
droplet generator produces droplets that are different in size from
those of a second droplet generator, the portions of the mold that
produce the first droplet generator, the second droplet generator,
or both, may be modified by a further processing step. Once the
mold has been modified to form modified mold 52', at least one
second copy (or many second copies) of the droplet-generating
device may be produced with mold 52', indicated at 104. The second
copy may be sold or may be used to produce emulsions, and the
droplet size (or other corresponding property) of the emulsions may
be determined as previously described. Accordingly, the mold may be
modified iteratively, any suitable number of times, by repeating
the steps of producing a droplet-generating device, generating sets
of droplets, and testing sets of droplets, until the mold does not
need further modification. Then, many copies of the
droplet-generating device can be manufactured with the same
mold.
In some embodiments, the mold may be constructed to create an array
of droplet generators that each generate droplets of the same
nominal size. However, since decreasing the size of generated
droplets through mold tuning may be preferred over increasing the
size of droplets, the initial nominal size may be slightly higher
than the final size desired. For example, the initial nominal size
may be at least about 0.5% or 1% greater than the final size
desired, to increase the probability that each droplet generator
produces droplets that are within the tolerance, or are above the
final size desired if outside the tolerance.
Further aspects of droplets, emulsions, droplet generation, droplet
generators, droplet-generating devices, producing
droplet-generating devices, detecting signals from droplets, and
assays performed with droplets, among others, are described in the
references identified above under Cross-References, which are
incorporated herein by reference.
II. Examples
The following examples describe selected aspects and embodiments of
the present disclosure related to methods of making a
droplet-generating device, methods of improving a mold for making a
molded portion of a droplet-generating device, and methods of
forming emulsions, among others. These examples are intended for
illustration only and should not limit or define the entire scope
of the present disclosure.
Example 1
Exemplary Droplet-Generating Device
This example describes an exemplary droplet-generating device 54
having a molded portion 58 that forms grooves of a microfluidic
layer of channels; see FIGS. 2-5.
FIGS. 2 and 3 show respective exploded and assembled views of
droplet-generating device 54, with molded portion 58
(interchangeably termed a substrate) separated from (FIG. 2) or
attached to (FIG. 3) a sealing member 120 that cooperatively forms
a microfluidic layer 122 of circumferentially-bounded channels with
the molded portion. The sealing member may form a fluid-tight seal
with molded portion 58 and may be attached to a surface 80 thereof.
Surface 80 may be a top surface, a bottom surface, or a lateral
side surface, among others, of the molded portion. The sealing
member may, for example, be a sheet of material, such as a film.
The sealing member may have a surface 124 that forms wall regions
of the microfluidic layer, such as a wall region of each channel.
In the depicted embodiment, a top surface of the sealing member
forms a floor region of each channel. In other embodiments, a
sealing member may be attached also or alternatively to the top
side of the molded portion to form an upper wall region (such as a
ceiling region) of each of a set of microfluidic channels.
Device 54 may include a plurality of integrally-formed emulsion
production units 130 (see FIGS. 2-5). (FIG. 4 shows device 54
inverted and with sealing member 120 removed.) Each unit 130 has a
droplet generator 82 formed by microfluidic layer 122 and
fluidically connected to reservoirs that each supply fluid to
and/or receive fluid from the droplet generator (see FIGS. 2, 4 and
5). The reservoirs may include at least one carrier well 132 and a
sample well 134 that respectively supply carrier fluid and sample
to the droplet generator (see FIGS. 3 and 5). The reservoirs also
may include an emulsion well 136 that receives sample droplets in
the carrier fluid from the droplet generator. In some embodiments,
the carrier well (or other reservoir) may be shared by two or more
droplet generators.
FIG. 5 shows an open channel structure 74 defined by molded portion
58 for one of units 130. Channel structure 74 includes a plurality
of grooves 76. The channel structure, when closed by sealing member
120, may form droplet generator 82 and a plurality of channels each
extending from one of wells 132, 134, or 136 to the droplet
generator. The channels may be input and output channels, namely,
at least one carrier channel, such as carrier channels 138a, 138b,
a sample channel 140, and a droplet channel 142. The carrier
channels direct carrier fluid to droplet generator 82, the sample
channel directs sample fluid to the droplet generator, and the
droplet channel directs droplets in the carrier fluid to the
emulsion well.
Flow in the channels can be induced by creating a pressure
differential. For example, a vacuum (negative pressure) can be
applied to the emulsion well, or positive pressure can be applied
to the sample and carrier wells.
Example 2
Exemplary Mold
This example describes an exemplary mold 52 for creating the molded
portion of the droplet-generating device of Example 1; see FIGS.
6-10.
The mold may be constructed with geometrical features that
correspond to the geometrical features desired in the molded
portion of the microfluidic device. The mold may be filled with a
fluid material. In the mold the fluid may be transformed to a solid
by thermal, chemical, and/or any other suitable process(es). Once
the material has solidified, the molded portion may be removed from
the mold.
FIG. 6 shows a sectional view of mold 52 prepared to receive fluid
material that will form the molded portion of the device 54. The
sectional view corresponds to the sectional view of an emulsion
production unit 130 in FIG. 3.
Mold 52 may include at least two parts that collectively define the
shape of at least a region of molded portion of device 54. One or
more or each of the parts may be described as a mold insert. The
parts may provide a channel-defining component 56 (interchangeably
termed a groove-defining component) and a well-defining component
150. The channel-defining component may create and be complementary
to a lower (or upper) region of the molded device portion, and the
well-defining component may create and be complementary to an upper
(or lower) region of the molded device portion, such as a majority
of each well. The mold may define a continuous void 152 that is
filled with material, such as a solidifiable fluid, during the
molding process. The void is bounded in part by ridges 68 that will
define grooves in the molded portion of device 54 (also see FIG.
1). The mold may be configured for an injection molding process to
create the molded portion of device 54.
The mold and the molded portion may have any suitable composition.
In exemplary embodiments, the mold is formed of metal and the
molded portion of a polymer (which may be described as a plastic).
An exemplary polymer that may be suitable is a cyclic olefin
polymer.
FIG. 7 shows channel-defining component 56 taken in isolation. The
component has a plurality of replicated projecting portions 62 each
including a ridge structure and each elevated from a planar surface
64 of a base 60. Each projecting portion may define a molded region
of an emulsion production unit 130 (see FIGS. 4 and 5). The
projecting portions may be discrete from one another, as shown
here, or may be continuous with one another. For example, the
projecting portions may create a continuous carrier channel that is
fluidically connected to each emulsion production unit. The
channel-defining component may have a larger footprint than molded
portion 58.
FIG. 8 shows one of projecting portions 62. The projecting portion
has a carrier platform 160, a sample platform 162, and an emulsion
platform 164 that create a bottom region of carrier well 132,
sample well 134, and emulsion well 136, respectively, in the
molding process (also see FIG. 3). Ridge structure 66 includes
ridges 68 that extend from and interconnect the three platforms,
and that form a junction region 71, also called a cross. Ridge
structure 66 is complementary to the open channel structure of the
molded portion of device 54 (compare with FIGS. 4 and 5). Ridges 68
include at least one carrier ridge, such as carrier ridges 166a and
166b, a sample ridge 168, and a droplet ridge 170.
FIG. 9 shows junction region 71 of projecting portion 62. A dashed
rectangle 180 identifies an exemplary region of the junction region
from which material may be removed (or added) to tune a droplet
generator molded in part by the junction region. The material may
be removed from any combination of junction 70, one or both carrier
ridges 166a, 166b, and/or emulsion ridge 170. In some embodiments,
material also or alternatively may be removed from sample ridge
168.
FIG. 10 shows a sectional view of junction region 71. A phantom
line 182 schematically illustrates where material may be removed
from the junction region to decrease the height of at least a
portion of the junction region, to tune a droplet generator molded
by the junction region. The material may be removed from a top side
of the junction region, which may reduce a height of the junction
region at one or more positions of the junction itself, and/or may
reduce a height across and/or along at least one ridge defined by
the mold. If removed from a ridge, the material may or may not be
removed uniformly in a direction across the ridge. For example,
material may be removed selectively from a transversely central
region of the top of the ridge relative to the lateral top edges of
the ridge, or vice versa.
Example 3
Exemplary Mold Modification
This example describes exemplary modification of the mold of
Example 2, and exemplary data collected after mold modification;
see FIGS. 11-13.
FIG. 11 shows a graph of height data collected from the top of
droplet ridge 170 of FIGS. 9 and 10, after removal of about 0.5
micron of material from boxed area 180 by irradiation with a laser.
The data was obtained by white light interferometry (WLI) with
scanning along the top of droplet ridge 170, on a straight line
extending through the points identified by a closed triangle 190
and an open triangle 192 in FIG. 9. An average change 194 in height
of 0.506 micron is measured.
FIG. 12 shows a graph with concentration data obtained from an
assay performed to test modification of a mold. A working model of
channel-defining component 56 of FIG. 7 first was constructed. Each
of the eight projecting portions 62, designated in order as
positions "1" through "8," were formed as substantially identical
replicates. Next, laser irradiation was conducted to remove 0.5
micron and 1.0 micron of material from the top side of the junction
region of two of the projecting portions (portions at positions "3"
and "5," respectively, of the eight positions), within the area
bounded by rectangle 180 of FIG. 9, to generate a modified mold.
The modified mold then was used to produce a working model of
droplet-generating device 54, with the mold creating molded portion
58 (see FIGS. 2-5) by defining the shape and shape of the molded
portion. Each of the eight projecting portions 62 of the modified
mold created a recessed region of one of the eight droplet
generators of device 54. (The droplet generators are numbered "1"
through "8" according to the position of the corresponding
projecting portion that created the recessed region of the droplet
generator.)
An emulsion was formed with each droplet generator from a separate
volume of the same sample. The sample contained a nucleic acid
target at a known concentration. The target was present at partial
occupancy in the droplets of each emulsion (i.e., a subset of the
droplets did not contain at least one copy of the target). The
target was amplified in droplets of each emulsion using the
polymerase chain reaction (PCR), in the presence of a
fluorophore-labeled probe for target amplification. Fluorescence
was detected from droplets of each emulsion, to determine the
fraction of droplets positive for amplification of the target. A
concentration (copies/droplet) of the target in each emulsion was
calculated using the target-positive fraction obtained for each
emulsion. The concentrations are graphed in FIG. 12 as the percent
shift in measured concentration from an expected concentration for
the nominal size of droplet. The droplet generators at positions
"3" and "5" exhibited the largest decrease in concentration,
because these droplet generators formed smaller droplets. The shift
in concentration and thus the decrease in droplet volume is
proportional to the depth of material removed.
FIG. 13 compares a theoretical ("calculated") concentration drop
expected for droplet generators "3" and "5" of FIG. 12, based on
the amount of material removed from the junction region ("the
cross"), with the experimentally determined drop in target
concentration observed for droplet generators "3" and "5" of FIG.
12. An algorithm that relates the amount (e.g., the depth) of
material removed from the junction region to the drop in
concentration (and thus the change in droplet volume) can be
derived from the calculated/experimental results of FIG. 13.
Accordingly, the amount of material to be removed from the junction
region to achieve a desired shift in droplet size can be determined
with the algorithm.
Example 4
Exemplary Parallel Droplet Generation for the Same Emulsion
This example describes an exemplary droplet-generating device 190
that may be produced with the methods of the present disclosure,
with the device utilizing two or more droplet generators 82 (e.g.,
generators 82a-82c) that operate in parallel to generate droplets
for the same emulsion; see FIG. 14.
Device 190 may include one or a plurality of emulsion production
units 130 that operate to form emulsions from a sample fluid and a
carrier fluid (e.g., oil). The sample fluid and the carrier fluid
may be held by a sample well 134 and a carrier well 132. Each well
may supply fluid to droplet generators 82a-82c. At least one
distinct sample channel 140 may extend from sample well 134 to each
droplet generator. A plurality of carrier channels 138 may be in
fluid communication with carrier well 132 and extend to the droplet
generators. (In the depicted embodiment, six carrier channels are
used, two for each droplet generator.) A droplet channel 142
extends from each droplet generator to the same emulsion well
136.
Carrier well 132 may communicate with carrier channels 138 via
vertical channels 200. The vertical channels allow the carrier well
to be vertically offset from sample channels 140, such that the
sample channels extend under (or over) the carrier well.
A mold that creates a molded portion of droplet generators 82a-82c
can be tuned according to the methods of the present disclosure to
provide a more uniform volume of droplets from the droplet
generators.
Example 5
Selected Embodiments
This example describes selected embodiments of a method of making a
droplet-generating device. The selected embodiments are presented
as a series of numbered paragraphs.
1. A method for making an improved mold for microfluidic devices is
disclosed, comprising the steps of: (A) making a mold; (B)
producing a molded microfluidic device comprising a plurality of
droplet generators using the mold; (C) generating a first set of
droplets using a first droplet generator; (D) generating a second
set of droplets using a second droplet generator; (E) determining a
property of the first set of droplets; (F) determining a property
of the second set of droplets; (G) determining a modification to
the mold based on the property of the first set of droplets, the
second set of droplets, or both; and (H) modifying the mold
according to the modification.
2. A method for making an improved mold for microfluidic devices is
disclosed, comprising the steps of: (A) making a mold; (B)
producing a molded microfluidic device comprising a plurality of
droplet generators using the mold; (C) generating a first set of
droplets using a first droplet generator; (D) determining a
property of the first set of droplets; (E) determining a
modification to the mold based on the property of the first set of
droplets; and (F) modifying the mold according to the
modification.
3. A method for making a microfluidic device is disclosed,
comprising the steps of: (A) making a mold according to paragraph 1
or paragraph 2; and (B) producing a molded microfluidic device
comprising a plurality of droplet generators using the mold.
4. A method for making an emulsion is disclosed, comprising the
steps of: (A) making a microfluidic device according to paragraph
3; and (B) producing an emulsion using the molded microfluidic
device.
5. A method of making a droplet-generating device, the method
comprising: (A) producing a first droplet-generating device
including a molded portion created at least in part with a mold and
also including a plurality of droplet generators each formed at
least in part by the molded portion; (B) generating a set of
droplets with each of one or more of the droplet generators; (C)
determining a property of at least one set of generated droplets;
(D) modifying the mold based on the property; and (E) producing a
second droplet-generating device including a molded portion created
at least in part with the modified mold.
6. The method of paragraph 5, wherein the step of determining a
property includes a step of determining an average number of copies
per droplet of a reference species in each set of generated
droplets.
7. The method of paragraph 6, wherein the reference species is a
nucleic acid target, and wherein only a subset of the droplets in
each set of generated droplets contain at least one copy of the
nucleic acid target.
8. The method of any of paragraphs 5 to 7, wherein the property
corresponds to a volume of individual droplets of the at least one
set.
9. The method of any of paragraphs 5 to 8, wherein the step of
generating a set of droplets includes a step of generating a
plurality of emulsions with the plurality of droplet generators,
with each emulsion containing portions of a same sample.
10. The method of any of paragraphs 5 to 9, wherein the molded
portion of the first droplet-generating device includes a surface
and a plurality of grooves formed in the surface, wherein each of
the plurality of droplet generators includes a channel intersection
at which a plurality of channels meet, and wherein each of the
plurality of channels is formed in part by one or more of the
plurality of grooves.
11. The method of paragraph 10, wherein the surface is present on a
top side or a bottom side of the molded portion of the first
droplet-generating device.
12. The method of paragraph 10, wherein the step of producing a
first droplet-generating device includes a step of attaching a
sealing member to the surface of the molded portion of the first
droplet-generating device to form circumferentially-bounded
channels from the plurality of grooves.
13. The method of any of paragraphs 5 to 12, wherein the step of
generating a set of droplets includes a step of generating a
distinct set of droplets with each of two or more of the droplet
generators, and wherein the step of determining a property includes
a step of determining a property of each set of droplets
generated.
14. The method of any of paragraphs 5 to 13, wherein the step of
determining a property includes a step of determining a property of
a distinct set of droplets generated by each of the plurality of
droplet generators.
15. The method of any of paragraphs 5 to 14, wherein the first
droplet-generating device has a first plurality of droplet
generators, wherein the second droplet-generating device has a
second plurality of droplet generators arranged in one-to-one
correspondence with the first plurality of droplet generators, and
wherein the step of modifying the mold causes the second plurality
of droplet generators to be configured to generate a more uniform
size of droplet than the first plurality of droplet generators.
16. The method of any of paragraphs 5 to 15, wherein the first
droplet-generating device has a first plurality of droplet
generators, wherein the second droplet-generating device has a
second plurality of droplet generators arranged in one-to-one
correspondence with the first plurality of droplet generators, and
wherein the step of modifying the mold causes at least one droplet
generator of the second plurality to be configured to generate a
smaller size of droplet than a corresponding droplet generator of
the first plurality.
17. The method of any of paragraphs 5 to 16, further comprising (a)
generating a set of droplets with each of one or more droplet
generators of the second droplet-generating device and (b)
determining a property of least one set of droplets generated with
the second droplet-generating device.
18. The method of paragraph 17, further comprising a step of
modifying the modified mold if the property of least one set of
droplets generated with the second droplet-generating device is
outside a tolerance.
19. The method of any of paragraphs 5 to 18, wherein the mold
defines a projecting portion including a plurality of intersecting
ridges that create a plurality of intersecting grooves in the
molded portion of the first droplet-generating device, and wherein
the step of modifying the mold includes a step of removing material
from the projecting portion of the mold.
20. The method of paragraph 19, wherein the projecting portion
includes a junction region having a junction where two or more of
the ridges meet one another, and wherein the step of removing
material includes a step of decreasing a height of the junction
region at one or more positions of the junction region.
21. The method of any of paragraphs 5 to 20, wherein the mold
defines a projecting portion including a plurality of intersecting
ridges that create a plurality of intersecting grooves in the
molded portion, wherein the step of modifying the mold includes a
step of changing a dimension of a region of the projecting portion,
and wherein the dimension is changed by less than one
micrometer.
22. The method of any of paragraphs 5 to 21, wherein the step of
modifying the mold is performed at least in part with a beam of
radiation.
23. The method of paragraph 22, wherein the step of modifying the
mold is performed by removal of material from the mold with a
laser.
24. A method of making a droplet-generating device, the method
comprising: (A) producing a first droplet-generating device
including a molded portion created at least in part with a mold and
defining a plurality of grooves and also including a plurality of
droplet generators each formed in part by an intersection of three
or more of the plurality of grooves; (B) generating a set of
droplets with each droplet generator; (C) determining an average
number of copies per droplet of a nucleic acid target in each set
of generated droplets; (D) modifying the mold based on the average
number of copies determined for one or more of the sets of
generated droplets; and (E) producing a second droplet-generating
device including a molded portion created at least in part with the
modified mold.
25. A method of making a droplet-generating device, the method
comprising: (A) producing a first copy of a droplet-generating
device including a molded portion created at least in part with a
mold and also including a plurality of droplet generators each
formed at least in part by the molded portion; (B) generating a set
of droplets with each of one or more of the droplet generators; (C)
determining a property of at least one set of generated droplets;
(D) modifying the mold based on the property; and (E) producing a
second copy of the droplet-generating device including a molded
portion created at least in part with the modified mold.
The disclosure set forth above may encompass multiple distinct
inventions with independent utility. Although each of these
inventions has been disclosed in its preferred form(s), the
specific embodiments thereof as disclosed and illustrated herein
are not to be considered in a limiting sense, because numerous
variations are possible. The subject matter of the inventions
includes all novel and nonobvious combinations and subcombinations
of the various elements, features, functions, and/or properties
disclosed herein. The following claims particularly point out
certain combinations and subcombinations regarded as novel and
nonobvious. Inventions embodied in other combinations and
subcombinations of features, functions, elements, and/or properties
may be claimed in applications claiming priority from this or a
related application. Such claims, whether directed to a different
invention or to the same invention, and whether broader, narrower,
equal, or different in scope to the original claims, also are
regarded as included within the subject matter of the inventions of
the present disclosure. Further, ordinal indicators, such as first,
second, or third, for identified elements are used to distinguish
between the elements, and do not indicate a particular position or
order of such elements, unless otherwise specifically stated.
* * * * *